Steven J. Feinmark

Attenuation of diet-induced atherosclerosis in rabbits with a highly selective 15-lipoxygenase inhibitor lacking significant antioxidant properties.

15‐Lipoxygenase (15‐LO) has been implicated in the pathogenesis of atherosclerosis because of its localization in lesions and the many biological activities exhibited by its products. To provide further evidence for a role of 15‐LO, the effects of PD 146176 on the development of atherosclerosis in cholesterol‐fed rabbits were assessed. This novel drug is a specific inhibitor of the enzyme in vitro and lacks significant non specific antioxidant properties. PD 146176 inhibited rabbit reticulocyte 15‐LO through a mixed noncompetitive mode with a Ki of 197 nm. The drug had minimal effects on either copper or 2,2′‐azobis(2‐amidinopropane)hydrochloride (ABAP) induced oxidation of LDL except at concentrations 2 orders higher than the Ki. Control New Zealand rabbits were fed a high‐fat diet containing 0.25% wt./wt. cholesterol; treated animals received inhibitor in this diet (175 mg kg−1, b.i.d.). Plasma concentrations of inhibitor were similar to the estimated Ki (197 nm). During the 12 week study, there were no significant differences in weight gain, haematocrit, plasma total cholesterol concentrations, or distribution of lipoprotein cholesterol. The drug plasma concentrations achieved in vivo did not inhibit low‐density lipoprotein (LDL) oxidation in vitro. Furthermore, LDL isolated from PD 146176‐treated animals was as susceptible as that from controls to oxidation ex vivo by either copper or ABAP. PD 146176 was very effective in suppressing atherogenesis, especially in the aortic arch where lesion coverage diminished from 15±4 to 0% (P<0.02); esterified cholesterol content was reduced from 2.1±0.7 to 0 μg mg−1 (P<0.02) in this region. Immunostainable lipid‐laden macrophages present in aortic intima of control animals were totally absent in the drug‐treated group. Results of these studies are consistent with a role for 15‐LO in atherogenesis.

Role of L-Type Calcium Channels in Pacing-Induced Short-Term and Long-Term Cardiac Memory in Canine Heart

Background—We tested the hypothesis that ICa,L is important to the development of cardiac memory. Methods and Results—The effects of L-type Ca2+ channel blockade and &bgr;-blockade were tested on acutely anesthetized and on chronically instrumented, conscious dogs. Short-term memory (STM) was induced by 2 hours of ventricular pacing and long-term memory (LTM) by ventricular pacing for 21 days. STM dogs received placebo, nifedipine, or propranolol, and LTM dogs received placebo, atenolol, or amlodipine. AT1 receptor blockade (candesartan) and ACE inhibition (trandolapril) were also tested in LTM. Microelectrodes were used to record transmembrane potentials from isolated epicardial and endocardial slabs using a protocol simulating STM in intact animals. Left ventricular epicardial myocytes from LTM or sham control dogs were dissociated, and ICa,L was recorded (whole-cell patch-clamp technique). Evolution of STM and LTM was attenuated by ICa,L blockers but not &bgr;-blockers. Neither AT1 receptor blockade nor ACE inhibition suppressed LTM. In microelectrode experiments, pacing induced an epicardial-endocardial gradient change mimicking STM that was suppressed by nifedipine. In patch-clamp experiments, peak ICa,L density in LTM and control were equivalent, but activation was more positive and time constants of inactivation longer in LTM (P <0.05). Conclusions—ICa,L blockade but not &bgr;-adrenergic blockade suppresses cardiac memory. LTM evolution is unaffected by angiotensin II blockade and is associated with altered ICa,L kinetics.

TREK-1 Is a Novel Molecular Target in Prostate Cancer

TREK-1 is a two-pore domain (K(2P)) potassium channel that carries a leak current that is time- and voltage-independent. Recently, potassium channels have been related to cell proliferation and some K(2P) family channels, such as TASK-3, have been shown to be overexpressed in specific neoplasms. In this study, we addressed the expression of TREK-1 in prostatic tissues and cell lines, and we have found that this potassium channel is highly expressed in prostate cancer but is not expressed in normal prostate nor in benign prostatic hyperplasia. Furthermore, expression of TREK-1 correlates strongly with the grade and the stage of the disease, suggesting a causal link between channel expression and abnormal cell proliferation. In vitro studies showed that TREK-1 is highly expressed in PC3 and LNCaP prostate cancer cell lines but is not detectable in normal prostate epithelial cells (NPE). In this report, we show that overexpression of TREK-1 in NPE and Chinese hamster ovary (CHO) cells leads to a significant increase in proliferation. Moreover, the increased cell proliferation rate of PC3 cells and TREK-1 overexpressing CHO cells could be reduced when TREK-1 current was reduced by overexpression of a dominant-negative TREK-1 mutant or when cells were exposed to a TREK-1 inhibitor. Taken together, these data suggest that TREK-1 expression is associated with abnormal cell proliferation and may be a novel marker for and a molecular target in prostate cancer.

Vascular smooth muscle cell leukotriene C4 synthesis: requirement for transcellular leukotriene A4 metabolism

Leukotriene synthesis and metabolism were studied in cultured porcine aortic smooth muscle cells (PSM). Cultures stimulated with calcium ionophore A23187, with or without exogenous arachidonic acid, did not release detectable levels of leukotriene B4, C4, D4 or E4. Those products were assayed by high-performance liquid chromatography, ultraviolet spectrometry and, in some cases, radioimmunoassay. Smooth muscle cultures were able to convert leukotriene A4 to leukotriene C4, indicating the presence of leukotriene C4 synthetase. Although this enzymatic activity has previously been found in cultured porcine aortic endothelial cells, it was not detectable in cardiac myocytes, fibroblasts from several organs or renal epithelial cells. It is known from previous work that inflammatory cells such as polymorphonuclear leukocytes (PMNL) or mast cells release leukotriene A4 when stimulated. Further, increased numbers of these cell-types are found associated with vascular tissue during several pathologic situations. Therefore, the potential for a leukocyte-smooth muscle cell interaction involving the transcellular metabolism of leukotriene A4 was assessed. Stimulation of PMNL suspensions in the presence of PSM resulted in a significant increase in total leukotriene C4 produced in comparison to either cell-type alone (255% of PMNL alone, P less than 0.05). Furthermore, after the intracellular glutathione pool of PSM was prelabelled with 35S, a PSM-PMNL coincubation produced levels of [35S]leukotriene C4 which were significantly greater (P less than 0.05) than those found after coincubating prelabelled PMNL with unlabelled PSM. These data demonstrate a PMNL-PSM interaction in which smooth muscle cell leukotriene C4 synthesis results from the transcellular metabolism of PMNL-derived leukotriene A4. Since leukotriene C4 and its metabolites are vasoconstrictors and cause increased vascular permeability, the biochemical interaction described in this report may be relevant to the pathophysiology of arterial vasospasm, atherogenesis and to the abnormalities of tissue perfusion associated with ischemic or inflammatory disorders.

Complement-independent Ab-induced peroxide lysis of platelets requires 12-lipoxygenase and a platelet NADPH oxidase pathway

Antiplatelet GPIIIa49-66 Ab of HIV-related thrombocytopenic patients induces thrombocytopenia and platelet fragmentation by the generation of peroxide and other reactive oxygen species (ROS). Here we report the presence of a functional platelet NADPH oxidase pathway that requires activation by the platelet 12-lipoxygenase (12-LO) pathway to fragment platelets. A new Ab-mediated mechanism is described in which the platelet 12-LO product, 12(S)-HETE activates the NADPH oxidase pathway to generate ROS.

Electrophysiologic Effects of Interactions Between Activated Canine Neutrophils and Cardiac Myocytes

Effects of Neutrophils on Cardiac Myocytes. Introduction: Myocardial ischemia causes neutrophils to bind to activaled myocytes and liberate platelet‐activating factor (PAF). PAF causes delayed repolarization, early afterdepolarizations (EADs), and arrest of repolarization. We studied the effect of activation of neutrophils bound to canine cardiac myocytes to determine if such activation causes PAF generation and similar changes in transmembrane potentials.

Arrhythmias Caused by Platelet Activating Factor

Platelet Activating Factor and Arrhythmias. Introduction: Both ischemia and reperfusion are associated with ventricular arrhythmias. In both instances, neutrophils migrate into the ischemic zone, are activated by locally released factors, and hind to myocytes. The activated neutrophils liberate platelet activating factor (PAF). We have studied the arrhythmogenic actions of PAF on transmembrane potentials of isolated canine cardiac myocytes.

Activation of Protein Kinase G Up-regulates Expression of 15-Lipoxygenase-1 in Human Colon Cancer Cells

Recent studies indicate that the induction of apoptosis in human colon cancer cells by certain nonsteroidal antiinflammatory drugs involves increased expression of 15-LOX-1 and synthesis of its major product 13-S-hydroxyoctadecadienoic acid (13-S-HODE). Evidence was obtained that this occurs via a cyclooxygenase-2 (COX-2)-independent mechanism, but the actual mechanism of induction of 15-LOX-1 by these compounds is not known. There is extensive evidence that treatment of SW480 human colon cancer cells with sulindac sulfone (Exisulind, Aptosyn) or the related derivative OSI-461, both of which inhibit cyclic GMP (cGMP)-phosphodiesterases but lack COX-2 inhibitory activity, causes an increase in intracellular levels of cGMP, thus activating protein kinase G (PKG), which then activates pathways that lead to apoptosis. Therefore, in the present study, we examined the effects of various agents that cause increased cellular levels of cGMP on the expression of 15-LOX-1 in SW480 human colon cancer cells. Treatment of the cells with Exisulind, sulindac sulfide, OSI-461, the guanylyl cyclase activator YC-1, or the cell-permeable cGMP compound 8-para-chlorophenylthio-cGMP (8-pCPT-cGMP) caused an increase in cellular levels of 15-LOX-1. Exisulind, OSI-461, and 8-pCPT-cGMP also increased mRNA levels of 15-LOX-1, suggesting that the effects were at the level of transcription. The cGMP-phosphodiesterase inhibitors and YC-1 increased the production of 13-S-HODE, which is the linoleic acid metabolite of 15-LOX-1. Treatment of SW480 cells with the PKG inhibitor Rp-8-pCPT-cGMP blocked Exisulind-induced 15-LOX-1 expression. Furthermore, derivatives of SW480 cells that were engineered to stably overexpress wild-type PKG Ibeta displayed increased cellular levels of 15-LOX-1 when compared with vector control cells. Taken together, these results provide evidence that the cGMP/PKG pathway can play an important role in the induction of 15-LOX-1 expression by nonsteroidal antiinflammatory drugs and related agents.

Interactions between antiarrhythmic drugs and cardiac memory.

OBJECTIVE Ventricular pacing or arrhythmias can induce cardiac memory (CM). We hypothesized that clinically administered antiarrhythmic drugs alter the expression of CM, and that the repolarization changes characteristic of CM can modulate the effects of antiarrhythmic drugs. METHODS We studied conscious, chronically-instrumented dogs paced for two 1-h periods to study the effects of drugs on the evolution of memory (protocol 1) or for 21 days (protocol 2) to observe the effects of steady-state memory on drug actions. Dogs were treated in both settings with quinidine, lidocaine or E4031, in random order, and within therapeutic serum concentration ranges. RESULTS Pacing, alone, for 2 h significantly prolonged ERP only near the left ventricular pacing site, whereas pacing alone for 21 days prolonged ERP at all sites (P<0.05). Quinidine and E4031, but not lidocaine, prolonged repolarization and ERP and suppressed evolution of CM in protocol 1. However, quinidines effect in prolonging repolarization was diminished in both protocols, while its effect in prolonging ERP was diminished in the 21-day protocol only. In contrast, the effects of E4031 were additive to those of CM, prolonging repolarization and ERP in both protocols, while lidocaine showed no changes in effect at all. CONCLUSIONS Pacing to induce CM significantly affects ventricular repolarization and refractoriness, and there are interactions between CM, quinidine and E4031. Depending on the specific drug, these interactions have the potential to be anti- or proarrhythmic, and may impact importantly on the clinical efficacy of drugs as well as on electrophysiologic testing of drug actions.

Is there a role for 15-lipoxygenase in atherogenesis?

15-Lipoxygenase has been suggested to play a role in atherogenesis. The proposed action of this enzyme is to oxidize low density lipoprotein (LDL) to the extent that LDL becomes a ligand for the macrophage scavenger receptor. 15-Lipoxygenase and oxidized LDL are co-localized in atherosclerotic lesions; antioxidant drugs that block the lipoxygenase also block oxidation of LDL and the progression of experimental atherosclerosis. Biochemical experiments have demonstrated that the lipoxygenase can be induced by cytokines and/or another factor(s) associated with hypercholesterolemia. However, molecular biological work has shown that induction of the enzyme alone is not sufficient to induce lesion formation. Furthermore, the mechanism of action of 15-lipoxygenase in atherogenesis remains unclear. Predictions of the stereochemistry of enzyme-oxidized linoleate products appear to conflict with the available data. In fact, most studies have discovered substantial levels of racemic 13-hydroxyoctadecadienoic acid (13-HODE) in arterial lesions rather than the stereochemically pure 13(S)-HODE expected from purified enzyme. The possibility that the generation of products of 15-lipoxygenase metabolism must occur in a specific cellular location and during a brief time window in the development of the disease has been discussed. It is also possible that the true function of the linoleate metabolites is to modulate gene expression and regulate mitogenesis, and that oxidation of LDL may play a secondary role. The advent of transgenic species that both develop atherosclerosis and either fail to express or overexpress the lipoxygenase presents an opportunity to clarify some of these issues in the near future.